GSM6590353: E. coli promoter activity, FACS, rep3 bin12; Escherichia coli str. K-12 substr. MG1655; OTHER
Reporter plasmids were purified from 3 mL aliquots of overnight cell cultures from each bin using miniprep kits. 50 ng purified plasmids for each bin were amplified by the KAPA HiFi PCR Kit (Kapa Biosystems) for 20 cycles using primers prYH071 and prYH087. The PCR products were purified using the DNA Clean & Concentrator kit, and then were ligated with Illumina sequencing adapters via Golden Gate assembly. The ligation products were gel-purified to select DNA size between 400 bp and 1.5 kb and were subjected to another PCR with primers prYH128 and prYH129 for 8 cycles by the KAPA HiFi to enrich ligated products. The samples from each binned population were mixed at an estimated ratio to ensure that the number of reads sequenced is approximately proportional to the number of cells sorted for each bin in each replicate experiment.